The long-term objective of Core C is to assess the in vitro efficacy and toxicity of Cyanovirin (generated by Lactobacilli) as well as the naturally occurring antimicrobial peptide, Retrocyclin. By providing a natural route of delivery for the microbicides and maintaining the acidic environment of the vagina, these microbicides offer a unique approach to HIV prevention. The proposed in vitro testing algorithm will aid in advancing safe, novel microbicide combination strategies from preclinical to clinical studies. Formulations of each microbicide will be tested in the individual laboratories for Project 1 (Cyanovirin) and Project 2 (Retrocyclin), and only those with the highest anti-HIV activity will be sent to Core C for testing. It is anticipated that one to two compounds per year from each project will be tested in the following in vitro algorithm: 1) Microbicides from Projects 1 and 2 will initially be tested in a CD4-dependent, cell-cell transmission assay to assess the ability of these compounds to inhibit cell to cell infection with a CCR5-tropic HIV-1 strain (JR-CSF molecular clone). 2) If positive activity is observed in this assay, testing of each active compound will be repeated in this assay in the presence of mucin or a pH transition. This secondary testing will help determine if interaction of the compound with mucin or an acidic environment, similar to what may occur in the vagina, might affect the ability of the microbicide to inihibit cell to cell HIV-1 transmission. 3) If positive activity is observed in the repeat testing, each active compound will be tested in a CD4-dependent, cell-cell transmission assay that utilizes a CXCR4-tropic HIV-1 strain (SK-1 clinical isolate); however, further testing with mucin or a pH transition will not be done for this assay. 4) In parallel to steps 1-3 of the algorithm, all compounds sent to this Core will be tested for toxicity by assessing their effect on L. crispatus and L. jensenii growth and determining those concentrations that inhibit bacterial growth. These data, in conjunction with tissue explant and animal studies, will help determine the best candidates for clinical trials.